EP3052111B1 - Zusammensetzungen mit einem bestimmten mikrobiom und verfahren zur verwendung davon - Google Patents

Zusammensetzungen mit einem bestimmten mikrobiom und verfahren zur verwendung davon Download PDF

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EP3052111B1
EP3052111B1 EP14851064.7A EP14851064A EP3052111B1 EP 3052111 B1 EP3052111 B1 EP 3052111B1 EP 14851064 A EP14851064 A EP 14851064A EP 3052111 B1 EP3052111 B1 EP 3052111B1
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lactobacillus
asf
bacteria
subject
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EP3052111A4 (de
EP3052111A1 (de
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Frederic Bushman
Gary Wu
James Lewis
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University of Pennsylvania Penn
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/375—Ascorbic acid, i.e. vitamin C; Salts thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A61K31/765—Polymers containing oxygen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/14—Peptides containing saccharide radicals; Derivatives thereof, e.g. bleomycin, phleomycin, muramylpeptides or vancomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones

Definitions

  • Hyperammonemia occurs in many inherited metabolic diseases, most prominently the urea cycle disorders in which there is a failure to detoxify ammonia to urea.
  • Current treatment includes a low-protein diet and drugs that relieve hyperammonemia, but these approaches are only partially effective in preventing/treating the devastating neurologic consequences caused by elevated levels of circulating ammonia.
  • a significant amount of body ammonia forms in the gut from the hydrolysis of urea by intestinal bacteria.
  • Oral antibiotic treatment may attenuate hyperammonemia, but the effectiveness of antibiotics wanes over time due to the development of antibiotic resistance.
  • WO2012/142605 discloses compositions comprising probiotic bacteria such as lactobacilli and bifidobacteria for the treatment of intestinal disorders.
  • a method for altering gut microbiota in a subject to reduce urease gene content or activity via reducing or eliminating urease producing bacteria from the gut in order to reduce ammonia production therefrom is disclosed.
  • a composition comprising a consortium of bacteria having no urease gene content or urease activity for use in a method of treating hyperammonemia, hepatic encephalopathy, or another liver disorder characterized by hyperammonemia in a subject.
  • the subject Prior to administration of said composition, the subject is given an effective amount of one or more antibiotics sufficient to reduce the population of bacteria in the gut microbiota to a level suitable for repopulation of newly introduced bacteria, and administered an effective amount of a purgative agent to the subject, thereby purging the intestines of said subject.
  • the method further comprises administering to the subject the bacteria having no urease activity or no urease gene content, under conditions wherein said bacteria colonize the gut.
  • the composition for use in the method of the invention comprises Paraprevotella clara, Bifidobacterium longum, Collinsella aerofaciens, Coprococcus comes, Dorea longicatena, Bacteroides eggerthii str., and Bacteroides vulgates bacteria.
  • the method results in the beneficial reduction of urease gene content, or activity or both in gut microbiota and ammonia production in the gut of the subject.
  • the subject has been treated with a purgative agent selected from the group consisting of polyethene glycol (PEG), magnesium citrate, sodium picosulphate plus magnesium citrate, PEG plus ascorbic acid, and sodium phosphate.
  • a purgative agent selected from the group consisting of polyethene glycol (PEG), magnesium citrate, sodium picosulphate plus magnesium citrate, PEG plus ascorbic acid, and sodium phosphate.
  • the intestinal purge is carried out for at least about 12-36 hours prior to the administration of the composition.
  • the subject has been administered one or more antibiotics for at least 48, 72, or 96 hours prior to administration of the composition, wherein the antibiotics are neomycin and vancomycin.
  • the subject has a disease is a metabolic disorder selected from the group consisting of Hyperammonemia, Transient Hyperammonemia of the Newborn or hepatic encephalopathy.
  • the composition can be administered to the subject via several routes, which include, without limitation, endoscopy, by enteroscopy, by colonoscopy, a nasoduodenal catheter, an enema and orally in a consumable capsule or pill.
  • the defined microbial consortia further comprises one or more bacteria belonging to a genus selected from the group consisting of Bifidobacterium, Bacteroides, Tannerella, Parabacteroides, Bacillus, Lactobacillus, Anaerostipes, Anaerostipes, Blautia, Coprococcus, Dorea, Clostridium XI, Collinsella, and Paraprevotella.
  • the consortia comprises Clostridium sp., Lactobacillus sp., Lactobacillus murinus, Mucispirillum schaedleri, Eubacterium plexicaudatum, Firmicutes bacterium, Clostridium sp.
  • the consortia consists of Paraprevotella clara, Bifidobacterium longum, Collinsella aerofaciens, Coprococcus comes, Dorea longicatena, Bacteroides eggerthii str., and Bacteroides vulgates.
  • the method comprises determining the efficacy of treatment by assessing a parameter selected from the group consisting of 16S rRNA copy number, 16S rRNA gene sequencing, fecal urease levels, fecal or circulating amino acids, biogenic amines, fecal ammonia levels, and circulating ammonia levels.
  • compositions comprising a defined microbial consortia of bacteria having no urease gene content or activity comprising Paraprevotella clara, Bifidobacterium longum, Collinsella aerofaciens, Coprococcus comes, Dorea longicatena, Bacteroides eggerthii str., and Bacteroides vulgates bacteria, said consortium being present in a biological carrier suitable for administration to, and colonization, of the gut.
  • An exemplary consortia further comprises at least one further bacterium having no urease gene content or activity selected from the group consisting of Bifidobacterium, Bacteroides, Tannerella, Parabacteroides, Bacillus, Lactobacillus, Anaerostipes, Anaerostipes, Blautia, Coprococcus, Dorea, and Clostridium XI.
  • the invention provides a composition comprising a single urease negative bacterial population for altering gut microbiota to reduce urease gene content or activity for use in a method of treating hyperammonemia, hepatic encephalopathy, or another liver disorder characterized by hyperammonemia in a subject.
  • the subject Prior to administration of the composition, the subject is given an effective amount of vancomycin and neomycin sufficient to reduce the population of bacteria in the gut microbiota to a level suitable for repopulation of newly introduced bacterium, and administered an effective amount of purgative agent.
  • the composition is then administered to the subject under confitions wherein said bacterium colonizes the gut.
  • the bacteria are selected from the group consisting of urease negative Lactobacillus acidophilus L. acidophilus, L. acidophilus, L. acidophilus, Lactobacillus casei, L. casei, Lactobacillus fermentum, Lactobacillus johnsonii Lactobacillus paracasei, Lactobacillus plantarum , Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus lactis, Lactobacillus dubrueckii bulgaricus, Bifidobacterium lactis, Bifidobacterium longum , Bifidobactenium breve, E. coli Nissle and Streptococcus thermopiles.
  • compositions comprise the bacteria species diluted in a biologically compatible solution, optionally containing excipients, suitable for administration to said subject.
  • a defined microbial consortia is meant a purified and/or isolated population of known microbes.
  • undesirable gut microbiome is meant a community of microbes comprising a pathogen or having a biological activity associated with a pathogenic process.
  • an undesirable gut microbiome comprises microbes having urease activity.
  • an undesirable gut microbiome comprises an increased number or percentage of Clostridium difficile relative to the number or percentage of C. difficile and/or toxin production present in the gut of a healthy control subject.
  • normal gut microbiota is meant a population of microbes that is substantially similar to the population of microbes present in the gut of a healthy control subject.
  • ameliorate decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
  • disease is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.
  • exemplary diseases include hyperammonemia, Clostridium difficile colitis, hepatic encephalopathy associated with cirrhosis, and inflammatory bowel disease.
  • an effective amount is meant the amount of a composition of the invention (e.g., defined microbial consortia) required to ameliorate the symptoms of a disease relative to an untreated patient.
  • the effective amount of a defined microbial consortia varies depending upon the manner of administration, the indication for administration, the age, body weight and height, sex, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.
  • isolated refers to material that is free to varying degrees from components which normally accompany it as found in its native state.
  • Isolate denotes a degree of separation from original source or surroundings.
  • Purify denotes a degree of separation that is higher than isolation.
  • a purified isolated bacterium that is part of a defined microbial consortia is at least about 90%, 95% or 100% free of bacteria, fungi, viruses, or other undefined microbes.
  • a marker of the invention is urease activity (e.g., fecal urease) or circulating ammonia.
  • reference is meant a standard or control condition.
  • subject is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
  • the term "about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
  • compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
  • the invention features use of a defined microbial consortia for the replacement of a gut microbiome associated with disease.
  • the invention is based, at least in part, on the discovery that transplantation of a defined microbial consortia into a subject provides for the long term replacement of a gut microbiome associated with disease. While fecal transplantation has been used to treat subjects with diseases associated with undesirable changes in the gut microbiome, inoculating subjects with undefined complex microbial communities raise a number of safety concerns. In particular, the possibility that fecal transplant might inadvertently result in the transfer of pathogens into a susceptible host. Therefore, a need exists to identify defined microbial consortia with well characterized biological functions that can be used to target gut microbiome-associated diseases.
  • fecal microbiota transplantation will be replaced by the use of laboratory generated resilient microbial consortia that can be used to target specific diseases based on well-characterized microbial biological properties.
  • FMT fecal microbiota transplantation
  • One such example is the deleterious effects of ammonia production by the gut microbiota on the central nervous system in patients with chronic liver disease and/or inborn errors of metabolism, an entity known as hepatic encephalopathy.
  • Treatments for hepatic encephalopathy include strategies to reduce microbial production of ammonia and its absorption. Therefore, the development of a resilient microbial community with minimal urease gene content to prevent or inhibit the production of ammonia should reduce the development of hepatic encephalopathy when inoculated into a susceptible patient population.
  • ASF has been used for decades to colonize immune deficient mice without evidence of deleterious effects. Nevertheless, metabolic profiling of both the host and the transplanted microbiota revealed a reduction in essential amino acids demonstrating the importance of microbial urease activity in maintenance of the syntrophic interaction between the gut microbiome and its host with respect to nitrogen balance.
  • the present invention provides methods of treating diseases or symptoms thereof associated with the presence of one or more undesirable bacteria in the gut of a subject. Accordingly, the invention provides compositions and methods for treating a subject having or at risk of developing a disease associated with undesirable changes in the gut microbiome, the method involving administering a therapeutically effective amount of a composition comprising a defined microbial consortia to a subject (e.g., a mammal, such as a mouse or human).
  • a subject e.g., a mammal, such as a mouse or human.
  • a method of treating a subject suffering from or susceptible to a metabolic disease e.g., an inborn error of metabolism, such as urea carbamylase deficiency.
  • the subject is identified as having a Urea Cycle Defect (e.g., Carbamyl phosphate synthetase deficiency, Ornithine transcarbamylase deficiency, Arginosuccinate synthetase deficiency, Arginosuccinate lyase deficiency, Arginase deficiency, or N-acetylglutamate synthetase deficiency.
  • the subject has an Organic Acidemias (e.g., Propionic acidemia, Methylmalonic acidemia, Isovaleric acidemia).
  • the disclosure provides methods wherein the subject has a Fatty Acid Oxidation Disorder (e.g., Short chain acyl-coA dehydrogenase deficiency, Medium-chain acyl-coA dehydrogenase deficiency, Long-chain acyl-coA dehydrogenase deficiency) or a disorder of Amino Acid Transport (e.g., Lysinuric protein intolerance, Hvperammonemia, hyperornithinemia, homocitrullinemia syndrome).
  • the subject has Transient Hyperammonemia of the Newborn, Hyperammonemia, hyperinsulinemi, or hypoglycemia syndrome.
  • the subject has cirrhosis, hepatic encephalopathy, or another liver disorder characterized by hyperammonemia.
  • the method includes the step of administering to the mammal a therapeutic amount of an amount of a composition comprising a defined microbial consortia (e.g., a community of microbes deficient in urease activity) sufficient to treat the disease or disorder or symptom thereof, under conditions such that the disease or disorder is treated.
  • a defined microbial consortia e.g., a community of microbes deficient in urease activity
  • compositions of the disclosure comprising a defined microbial consortia preferably contain a combination of one or more (e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50) bacterial strains listed in Tables 1, 2, or 3A, 3B, 3C, 7A or 7B.
  • Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides hASF, common in healthy gut populate the nodes Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Petrimortas Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Tannerella common in healthy gut, populate the nodes Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Para bacteroides hASF, common in healthy gut.
  • probiotic lineage Firmicutes Clostridia Ciostridiales Lachnospiraceae Anaerostipes common in healthy gut populate the nodes Firmicutes Clostridia Clostridiales Lachnospiraceae Blautia common in healthy gut, populate the nodes Firmicutes Clostridia Ciostridiales Lachnospiraceae Coprococcus common in healthy gut, populate the nodes Firmicutes Clostridia Clostridiales Lachnospiraceae Dorea common in healthy gut, populate the nodes Firmicutes Clostridia Clostridiales Lachnospiraceae Oribacterium Firmicutes Clostridia Clostridiales Lachnospiraceae Syntrophococcus Firmicutes Clostridia Clostridiales Peptostreptococcaceae Clostridium XI hASF, common in healthy gut, populate the nodes Firmicutes
  • Table 3A Engineered human community low in urease Paraprevotella clara Bifidobacterium longum Collinsella aerofaciens Coprococcus comes Dorea longicatena Bacteroides eggerthii str. Bacteroides vulgatus Table 3B Human orthologs of ASF Clostridium sp. 356 Lactobacillus sp. 360 Lactobacillus murinus 361 Flexistipes group 457 Eubacterium plexicaudatum 492 Low G--C content Gram + group 500 Clostridium sp. 502 Bacteroides sp. 519 Table 3C Mouse ASF species TABLE 3C Genome features and accession numbers of the ASF bacteria PMID: 24723722 ASF no.
  • Taxonomy Genome size (Mb) GC (%) Gene count Contig count N 50 (kb) Fold coverage GenBank accession no. ASF356 Clostridium sp. 2.91 30.91 2,799 31 209 143 AQFQ00000000.1 ASF360 Lactobacillus sp.
  • Identifying a subject in need of treatment for a disease associated with the gut microbiome can be in the judgment of a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).
  • the terms "treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
  • the terms "prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of, or susceptible to, developing a disorder or condition.
  • the therapeutic methods of the invention in general comprise administration of a therapeutically effective amount of a composition comprising a defined microbial consortia to a subject (e.g., human) in need thereof.
  • a subject e.g., human
  • Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of those subjects "at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider.
  • a subject is effectively treated whenever a clinically beneficial result ensues. This may mean, for example, a complete resolution of the symptoms associated with an undesirable gut microbiota, a decrease in the severity of the symptoms associated with an undesirable gut microbiota, or a slowing of the progression of symptoms associated with an undesirable gut microbiota.
  • symptoms can be the result of an inborn metabolic error, for example, a urease cycle deficiency; a liver injury or chronic liver disease.
  • such patients have an impaired ability to process the ammonia delivered to the liver from the intestinal tract.
  • high circulating ammonia levels hypererammonemia
  • the disclosed methods include administering a defined microbial consortia having reduced urease activity to a patient having an undesirable gut microbiota.
  • These methods can further include the steps of a) identifying a subject (e.g., a patient and, more specifically, a human patient) who has an undesirable gut microbiota, and b) providing to the subject a composition comprising a defined microbial consortia described herein.
  • a composition comprising a defined microbial consortia described herein.
  • An amount of such a composition provided to the subject that results in a complete resolution of the symptoms associated with an undesirable gut microbiota, a decrease in the severity of the symptoms associated with an undesirable gut microbiota, or a slowing of the progression of symptoms associated with an undesirable gut microbiota is considered a therapeutically effective amount.
  • the present methods may also include a monitoring step to help optimize dosing and scheduling as well as predict outcome.
  • the disclosed methods can include the steps of administering a treatment to reduce the level of bacteria within the intestinal lumen of the subject prior to administering a composition comprising any of the defined microbial consortia described herein.
  • a treatment to reduce the level of bacteria within the intestinal lumen of the subject prior to administering a composition comprising any of the defined microbial consortia described herein.
  • Pretreatment regimens can include specific antimicrobial agents, for example one or more antibiotics, for example, rifaximin, metronidazole, trimethoprim-sulfamethoxazole, neomycin or vancomycin.
  • Pretreatment regimens of the disclosure can include non-specific agents, for example, a purgative, for example, polyethylene glycol or a laxative, for example, bisacodyl.
  • Pretreatment regimens of the disclosure can also include dietary modification.
  • the pretreatment regimens of the disclosure can include a combination of any of the treatment modalities above, for example, administration of an antibiotic, a purgative, a laxative, along with dietary modification.
  • Concurrent administration of two or more agents does not require that the agents be administered at the same time or by the same route, as long as there is an overlap in the time period during which the agents are exerting their therapeutic effect. Simultaneous or sequential administration is contemplated, as is administration on different days or weeks.
  • compositions of the invention can also include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial, isotonic and absorption delaying agents, buffers, excipients, binders, lubricants, gels, surfactants and the like, that may be used as media for a pharmaceutically acceptable substance.
  • the methods of the invention include administration of a defined microbial consortia of bacteria having minimal urease activity formulated as pharmaceutical compositions which contain, as the active ingredient, the defined microbial consortia described herein, in combination with one or more pharmaceutically acceptable carriers.
  • the defined microbial consortia can be sterilized using conventional sterilization techniques before or after it is combined with the pharmaceutically acceptable carrier.
  • the defined microbial consortia are typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, for example, a capsule, tablet, sachet, paper, or other container.
  • the excipient when it serves as a diluent, it can be a solid, semisolid, or liquid material (e.g., normal saline), which acts as a vehicle, carrier or medium for the active ingredient.
  • the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.
  • the type of diluent can vary depending upon the intended route of administration.
  • compositions can include additional agents, such as preservatives.
  • the excipient or carrier is selected on the basis of the mode and route of administration. Suitable pharmaceutical carriers, as well as pharmaceutical necessities for use in pharmaceutical formulations, are described in Remington's Pharmaceutical Sciences (E. W. Martin), a well-known reference text in this field, and in the USP/NF (United States Pharmacopeia and the National Formulary).
  • excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose.
  • the formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents.
  • the pharmaceutical compositions can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.
  • compositions for use in the present methods can be entrapped in a colloid for oral delivery.
  • Pharmaceutically acceptable compositions can be prepared according to standard techniques.
  • the defined microbial consortium having minimal urease activity can be dried and compacted by grinding or pulverizing and inserted into a capsule for oral administration.
  • the defined microbial consortium having minimal urease activity can be combined one or more excipients, for example, a disintegrant, a filler, a glidant, or a preservative.
  • Suitable capsules include both hard shell capsules or soft-shelled capsules. Any lipid-based or polymer-based colloid may be used to form the capusule.
  • Exemplary polymers useful for colloid preparations include gelatin, plant polysaccharides or their derivatives such as carrageenans and modified forms of starch and cellulose, e.g., hypromellose.
  • other ingredients may be added to the gelling agent solution, for example plasticizers such as glycerin and/or sorbitol to decrease the capsule's hardness, coloring agents, preservatives, disintegrants, lubricants and surface treatment.
  • the capsule does not include gelatin.
  • the capsule does not include plant polysaccharides or their derivatives.
  • compositions comprising the defined microbial consortium having minimal urease activity can be formulated in accordance with their use.
  • These compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be oral, gastrointestinal, or rectal. Administration can be in the form of a single bolus dose, or may be, for example, by a continuous perfusion pump.
  • compositions can be formulated in a unit dosage form, each dosage containing, for example, from about 0.005 mg to about 2000 mg of a defined microbial consortium having minimal urease activity per dose.
  • unit dosage forms refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
  • the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention.
  • the active ingredient is typically dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.
  • This solid preformulation is then subdivided into unit dosage forms of the type described above containing from, for example, 0.005 mg to about 1000 mg of the defined microbial consortium having minimal urease activity of the present invention.
  • compositions can be formulated in a unit dosage form, each dosage containing, for example, from about 0.1 mg to about 50 mg, from about 0.1 mg to about 40 mg, from about 0.1 mg to about 20 mg, from about 0.1 mg to about 10 mg, from about 0.2 mg to about 20 mg, from about 0.3 mg to about 15 mg, from about 0.4 mg to about 10 mg, from about 0.5 mg to about 1 mg; from about 0.5 mg to about 100 mg, from about 0.5 mg to about 50 mg, from about 0.5 mg to about 30 mg, from about 0.5 mg to about 20 mg, from about 0.5 mg to about 10 mg, from about 0.5 mg to about 5 mg; from about 1 mg from to about 50 mg, from about 1 mg to about 30 mg,, from about 1 mg to about 20 mg, from about 1 mg to about 10 mg, from about 1 mg to about 5 mg; from about 5 mg to about 50 mg, from about 1 mg to about 30 mg, from about 1 mg to about 20 mg, from about 1 mg to about 10 mg, from about 1 mg to about 5 mg; from
  • tablets or pills of the present invention can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
  • the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former.
  • the two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release.
  • enteric layers or coatings such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.
  • compositions of the invention in a pharmaceutical composition can vary depending upon a number of factors including dosage, chemical characteristics (e.g., hydrophobicity), and the route of administration.
  • dosage can be provided in a capsule containing from about 0.005 mg to about 1000 mg for oral administration.
  • dosage can be expressed as cfu/g of dry weight.
  • the dosage may vary, but can range from the equivalent of about 10 2 to about 10 12 cfu/g, e.g., 1 x10 2 cfu/g, 5 x10 2 cfu/g, 1 x10 3 cfu/g, 5 x10 3 cfu/g, 1 x10 4 cfu/g, 5 x10 4 cfu/g, 1 x105 cfu/g, 5 x10 5 cfu/g, 1 x10 6 cfu/g, 5 x10 6 cfu/g, 1 x10 7 cfu/g, 5 x10 7 cfu/g, 1 x10 8 cfu/g, 5 x10 8 cfu/g, 1 x10 9 cfu/g, 5 x10 9 cfu/g, 1 x10 10 cfu/g, 5 x10 10 cfu/g, 1 x10 11 c
  • compositions comprising a defined microbial consortia can be administered to the gastrointestinal tract of a subject by nasoduodenal catheter, by enema, or by endoscopy, enteroscopy, or colonoscopy or orally in a consumable capsule or pill.
  • the defined microbial consortia are diluted in a suitable excipient (e.g., saline solution).
  • the bacteria are delivered in lyophilized form.
  • the dosage required will depend on the route of administration, the nature of the formulation, the nature of the subject's condition, e.g., immaturity of the immune system or a gastrointestinal disorder, the subject's size, weight, surface area, age, and sex, other drugs being administered, and the judgment of the attending clinicians.
  • suitable dosages are in the range of 0.01-1,000 mg/kg. Some typical dose ranges are from about 1 ⁇ g/kg to about 1 g/kg of body weight per day. In some embodiments, the dose range is from about 0.01 mg/kg to about 100 mg/kg of body weight per day.
  • the dose can be, for example, 1 mg/kg, 2 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 50 mg/kg or 100 mg/kg.
  • the dosage can be expressed as cfu/g of dry weight.
  • the dosage may vary, but can range from the equivalent of about 10 2 to about 10 12 cfu/g, e.g., 1 x10 2 cfu/g, 5 x10 2 cfu/g, 1 x10 3 cfu/g, 5 x10 3 cfu/g, 1 x10 4 cfu/g, 5 x10 4 cfu/g, 1 x10 5 cfu/g, 5 x10 5 cfu/g, 1 x10 6 cfu/g, 5 x10 6 cfu/g, 1 x10 7 cfu/g, 5 x10 7 cfu/g, 1 x10 8 cfu/g, 5 x10 8 cfu/g, 1 x10 9 cfu/g, 5 x10 9 cfu/g, 1 x10 10 cfu/g, 5 x10 10 cfu/g, 1 x10 11
  • the dosage is likely to depend on such variables as the type and extent of progression of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the compound selected, formulation of the excipient, and its route of administration.
  • Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • Useful assay systems include for example, analysis of the particular gut microbial community, fecal urease activity or ammonia levels. Wide variations in the needed dosage are to be expected in view of the variety of useful microbial consortia and the differing efficiencies of various routes of administration. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art.
  • Administrations can be single or multiple (e.g., 2- or 3-, 4-, 6-, 8-, 10-, 20-, 50-, 100-, 150-, or more fold).
  • the duration of treatment with any composition provided herein can be any length of time from as short as one day to as long as the life span of the host (e.g., many years).
  • a composition can be administered once a week (for, for example, 4 weeks to many months or years); once a month (for example, three to twelve months or for many years); or once a year for a period of 5 years, ten years, or longer.
  • the frequency of treatment can be variable.
  • the present compositions can be administered once (or twice, three times, etc.) daily, weekly, monthly, or yearly.
  • a subject can be monitored for symptomatic relief, e.g., relief from lethargy, irritability, poor feeding, vomiting hyperventilation, seizures, ataxia.
  • serum markers for example, plasma concentration of ammonium, arginine, citrulline; urinary markers, for example, orotic acid can also be assayed
  • compositions of the disclosure may also be administered in conjunction with other therapeutic modalities.
  • Other therapeutic modalities will vary according to the particular disorder, but can include, for example, dietary modification, hemodialysis, therapeutic agents such as sodium benzoate, phenylacetate, arginine, or surgical remedies, for example, liver transplantation.
  • Concurrent administration of two or more therapeutic agents does not require that the agents be administered at the same time or by the same route, as long as there is an overlap in the time period during which the agents are exerting their therapeutic effect. Simultaneous or sequential administration is contemplated, as is administration on different days or weeks.
  • the invention provides a method of monitoring treatment progress, for example, by measuring urease activity (e.g., fecal urease), fecal ammonia levels or circulating ammonia levels.
  • urease activity e.g., fecal urease
  • the monitoring involves detecting gut microbes (e.g., by 16S rRNA gene sequencing or 16S rRNA copy number).
  • the method includes the step of determining a level of diagnostic marker (marker) or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to a disorder or symptoms thereof associated with dysbiosis, in which the subject has been administered a therapeutic amount of a composition herein sufficient to treat the disease or symptoms thereof.
  • the level of marker determined in the method can be compared to known levels of marker in either healthy normal controls or in other afflicted patients to establish the subject's disease status.
  • a second level of marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy.
  • a pre-treatment level of marker in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of marker can then be compared to the level of marker in the subject after the treatment commences, to determine the efficacy of the treatment.
  • a level of marker in the subject is determined serially after beginning treatment according to this invention; the level of marker can then be compared over time to determine the efficacy of the treatment and need for dose adjustment.
  • compositions comprising a defined microbial consortia are preferably administered to a subject by nasoduodenal catheter, by enema, or by endoscopy, enteroscopy, or colonoscopy or orally in a consumable capsule or pill.
  • the defined microbial consortia are diluted in a suitable excipient (e.g., saline solution).
  • the bacteria are delivery in lyophilized form.
  • the microbial consortium will be delivered as a lyophilized (freeze-dried) powder packaged in a consumable gelatin capsule.
  • the liquid culture will be: centrifuged, resuspended in a lyophilization medium which will optionally include cryoprotectants and biological- and/or chemical-oxygen scavengers (See Table 5) transferred under anaerobic conditions to a shelf lyophilizer, lyophilized, encapsulated in a gelatin capsule under anaerobic conditions, and packaged in a glass ampoule to maintain oxygen free conditions during transport and storage. Details on each of these steps, as well as method development and quality control (QC) considerations are detailed below.
  • Liquid cultures (10 10 colony forming units, or CFU) of the strain(s) in the logarithmic or early stationary phase of growth are centrifuged to form a pellet of cells.
  • the culture medium (supernatant) is then removed, and the pellet resuspended in a lyophilization medium; multiple lyophilization medium can be utilized and include without limitation, those set forth in (Table 1).
  • Table 4 Different lyophilization medium for formulation of the microbial consortium.
  • Lyophilization Medium 10-20% (wt./vol) skim milk 1-3 Reagent 18 (Tryticase Soy Broth, Sucrose, Bovine Serum Albumin Fraction V) 1,2 Reagent 20 (Sucrose, Bovine Serum Albumin Fraction V) 1 Microbial Freeze Drying Buffer 2,3
  • oxygen-scavenging excipients for reducing O 2 , thereby protecting the obligate anaerobic consortium (Tables 2 and 3A, 3B, 3C, 7A and 7B).
  • Chemical excipients include, without limitation, (1) ferrous sulfide (125 ⁇ M) 1,4,5 and (2) L-cysteine HCl (10 mM) 6,7 ; additionally, (3) Lactobacillus acidophilus (10 10 CFU), a facultative anaerobe widely used in probiotics 8,9 , will be used as a biological excipient both with and without the chemical excipients.
  • freeze-drying indicator OPS Diagnostics, Lebanon, NJ
  • OPS Diagnostics a colorimetric indicator that turns from red to blue when the water content is below 2%
  • the solution containing the lyophilization medium, resuspended pellet, and excipient(s) will be transferred to a lyophilizer that has been flushed with nitrogen gas to maintain an anaerobic headspace.
  • Lyophilizers are commercially available and can be obtained from BenchTop Pro with Omnitronics, SP Scientific, Warminster, PA for example. Lyophilization will initially be perfomed using the steps set forth below and in the following references: ( ATCC. Methods for freezing and freeze-dryingbacteria. 2014. Available on the world wide web at atcc.custhelp.com/app/answers/detail/a_id/140/ ⁇ /methods-for-freezing-and-freeze-dryingbacteria ; Diagnostics O.
  • Bacteria Freeze Drying Protocol 2014. Available at: on the world wide web at.opsdiagnostics.com/notes/ranpri/rpbacteriafdprotocol.htm ; Diagnostics O. Bacteria Lyophilization Overview. 2014. Available at: on the world wide web at http://opsdiagnostics.com/ notes/ranpri/bacteria_lyophilization_overview.htm ; Savini M, et al. Nutrients 2010;2(3):330-9 ;. Gitaitis RD. Refinement of Lyophilization Methodology for Storage of Large Numbers of Bacterial Strains. Plant Dis. 1987;July:615 ; Simon EM, et al. Maintaining Cultures for Biotechnology and Industry.
  • Cells are grown to late log or early stationary phase under optimal conditions on the medium of choice, harvested by centrifugation and the culture broth removed. The pellet is then resuspended in an in equal volume of lyophilization medium. Cells are divided into aliquots and placed into sterile vials or tubes (approximately 250-500 ⁇ l representing ⁇ 10 8 bacteria). The tubes are then placed in the lyophilizer for a suitable time period to ensure full lyophilization of the sample. The samples are frozen down to -40°C.When using instruments that provide for control of rate of freezing, a drop of 1°C per minute is preferred. Once the samples reach temperature, they should be visibly frozen. The sample is then subjected to vacuum pressure for drying.
  • Primary drying is the longest phase of the freeze drying process. The time for primary drying will also depend upon the volume of the sample. For bacteria, samples rarely need to be large and typically are 0.25 to 0.5 ml. A limited number of samples (10-20) in a shelf dryer can be completely dried in just a couple of hours. A fully loaded dryer with several hundred samples will take longer. As a standard guide, freeze dry overnight.
  • Samples may still contain moisture following primary drying. The amount is variable but ranges between 2 and 4%. This moisture level can be reduced by pumping heat into the sample during the secondary drying phase. This phase is relatively short, lasting 1 to 2 hours, but important for long-term viability.
  • the vials are stoppered using the stoppering plate/mechanism. The vacuum is then released, the vials removed and the stoppers further secured with rubber bungs/stoppers with foil crimp seals. It is best to store the vials at 4°C in the dark.
  • freeze dried bacteria are tested for viability as compared to the original culture and can be monitored for stability/viability of the freeze dried cultures by testing at 30, 90, 180 and 365 days.
  • the resulting product will be then be transferred back to the anaerobic chamber for downstream processing.
  • the lyophilized product will be weighed (to ensure 10 10 CFU/capsule) and packaged in gelatin capsules (Capsugel, Morristown, NJ) under anaerobic conditions. These capsules will then be packaged in glass ampoules (Schott, Elmsford, NY) to maintain anaerobic conditions during shipment and storage.
  • Validation will be performed by breaking the ampoule under aerobic conditions (as would be encountered when delivering the capsule to a subject in a medical setting) and then placing the gel capsule in prereduced culture medium (same medium as was used to grow the initial liquid cultures). Growth can be measured using optical density (OD 600 ) and colony counting (CFU/mL) at six hour time points up to three days after inoculation. This will ensure viability of the consortium for delivery to a human subject via this method over a continuous five day dosage period.
  • OD 600 optical density
  • CFU/mL colony counting
  • Reduction in the concentration of bacteria within the gut lumen prior to the administration of microbial consortium is accomplished with a combination of antibiotics that are effective within the intestinal lumen, dietary modification and a bowel lavage to reduce the fecal biomass.
  • adult patients will consume vancomycin 500mg orally every 6 hours and neomycin 1000mg orally every 6 hours.
  • the dose for children must be reduced according to the child's weight (vancomycin 40 mg/kg/day in 4 divided doses, not to exceed 500 mg PO every 6 hours; neomycin 25 mg/kg/dose PO every 8 hours, not to exceed 1 gram PO every 8 hours).
  • vancomycin 40 mg/kg/day in 4 divided doses not to exceed 500 mg PO every 6 hours; neomycin 25 mg/kg/dose PO every 8 hours, not to exceed 1 gram PO every 8 hours.
  • polyethylene glycol based bowel purgative such as GoLytely®
  • Slight variation in the preparation regimen is possible, particularly with respect to the timing of administration of the polyethylene glycol based bowel purge. This can be administered on either the 2 nd or the 3 rd day of the regimen.
  • smaller volume polyethylene glycol bowel purgatives can be combined with oral laxatives such as bisacodyl.
  • the microbial consortium will be inoculated either orally via a capsule, into the upper GI track via a nasoduodenal tube, endoscopy or enteroscopy or into the lower GI tract via a colonoscopy or enema.
  • the inoculation will be a minimum of one day but may be repeated daily for a maximum of 7 days.
  • the preparation regimen is not required for chronic dosing of microbial consortium. However, following a lapse in therapy the bowel preparation regimen may be required prior to re-initiation of therapy.
  • kits for promoting the expansion of a defined microbial consortia in the gut of a host comprises a sterile container which contains a therapeutic or prophylactic composition comprising the defined microbial consortia; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
  • the kit may also contain the gelatin capsules described above and instructions for methods of administration.
  • the kit preferably contains instructions that generally include information about the use of the composition for the expansion of the microbial consortia in the gut of the subject.
  • the kit further contains precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references.
  • the instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
  • Example 1 ASF is a distinctive gut microbial community that exhibited resiliency under SPF housing conditions.
  • ASF consists of 8 bacterial strains developed in the 1970s that are collectively known to induce immune tolerance, and are currently used commercially to colonize immune deficient mice to maintain health.
  • 16S rRNA gene sequencing was performed on fecal DNA obtained from ASF colonized mice housed in an isolator.
  • Figure 1A the ASF community could be visualized as a very distinctive community on a UniFrac-based PCoA analysis ( Figure 1B ).
  • Example 2 Preparation of mice with antibiotics and polyethylene glycol allows the transplantation of ASF into previously colonized mice.
  • Example 3 Long-term taxonomic resilience of the transplanted ASF community established by the ability of Parabacteroides to inhibit invasion of Bacteroides, but not Clostridia genera into the gut microbiota niche.
  • the bacteria in the Bacteroides genus are the dominant taxa in conventionally housed mice.
  • ASF community persists, but specific organisms from the pretransplant community slowly increase in abundance ( Figure 4B ).
  • the bacterial taxa capable of time-dependent displacement of the ASF community were specific taxa primarily from the Clostridium genus.
  • Parabacteroides maintained its niche in the gut as the predominant representative of the Bacteroidetes phylum, but an equilibrium was established with the Firmicutes phylum primarily composed of the Clostridium genus.
  • Example 4 ASF has minimal urease gene representation and enzymatic activity.
  • Example 5 The ASF community and transplanted mice are both deficient in essential amino acids.
  • aromatic amino acids are precursors to biogenic amines that are neurotransmitters such as catecholamines and serotonin as well as false neurotransmitters including phenylethanolamine, octopamine, and synephrine.
  • Normally aromatic amino acids are decarboxylated to their respective amines, which subsequently undergo additional metabolism in the liver by monoamine oxidase into aldehydes and ammonia.
  • the precursor aromatic amino acids and their amines are shunted away from the pathway of aldehyde formation and may enter the central nervous system to become locally B-hydroxylated into false neurotransmitters and replace normal neurotransmitters 20 .
  • BCAA/AAA (valine) + (leucine) + (isoleucine) / (phenylalanine) + (tyrosine)
  • ASF colonization led to a remarkable alteration in plasma levels of essential amino acids with a significantly higher Fischer's Ratio (3.87) relative to mice with a conventional microbiota (2.68, p ⁇ 0.01).
  • Example 6 ASF transplantation reduced cognitive impairment and mortality in a murine model of acute liver injury.
  • HE hepatic encephalopathy
  • mice In mice, the TAA model has also been associated neurobehavioral abnormalities resembling hepatic encephalopathy in humans (24).
  • TAA neurobehavioral abnormalities resembling hepatic encephalopathy in humans.
  • NF normal feces
  • TAA treated mice transplanted with NF showed a decrease in cognitive function, quantified as spontaneous alternations in a Y-maze test, whereas ASF treated mice were not different from untreated controls (( Fig. 10b )).
  • a possible alternative could be that ASF transplantation directly reduced liver injury.
  • ALT plasma alanine aminotransferase
  • Fig. 11a and Fig. 11b histologic evidence of hepatocyte necrosis
  • FMT FMT in the treatment of Clostridial difficile infection supports the concept of a resilient microbial community that, when transferred into a host with disease, can be therapeutic by altering a dysbiotic microbiota.
  • feces contains not only bacteria but also a multitude of archaeal, fungi, and viruses that have been poorly characterized and can change in ways that cannot be predicted, there is significant concern for both short- and long-term risks of FMT in humans.
  • FMT refractory Clostridial difficile infections
  • ASF as a well-characterized, innocuous microbial community with beneficial immunologic properties provided evidence of efficient transfer into previously a colonized host and model both its taxonomic and functional resiliency over time with the host in a SPF environment.
  • composition of ASF has been recently described with greater precision based on 16S rRNA gene phylotyping.
  • the ASF community remains relatively stable with Parabacteroides remaining the dominant taxon over one month in SPF house mice.
  • the succession of ASF engraftment can be evaluated by the examination of the early time points during and after the inoculation of ASF.
  • the lactobacilli genus is present in most mice prior to transfer but a number of the low abundance ASF taxa, Mucispirilum, Oscilospria, and Lachnospariceae, appear with repetitive gavage in both germ free and pre-treated conventional hosts. The last to appear is the Clostridial genus at day 14 in the latter hosts, one week after gavaging was complete perhaps indicating the need for the establishment of a specific niche that permits the establishment of more fastidious taxa.
  • Species rich communities tend to exhibit enhanced resiliency that are less prone to invasion by new species possibly because limiting resources are used more efficiently with different species specialized to each potentially limiting resource ( Yatsunenko T et al., 2012, Nature, 486.7402: 222-227 ). In part, this may be due to the functional redundancy of a rich microbial community whereby multiple rare species are capable to filling a niche when an abundant species is compromised by an environmental disturbance ( Yatsunenko T et al., 2012, Nature, 486.7402: 222-227 ).
  • the bacterial taxa that invade the gut niche of ASF colonized mice are time dependent, consistent between all three models, and almost exclusively involve the Clostridium genus collectively reducing the proportional abundance of Parabacteroides by about 50%.
  • the relative proportion of Parabacteroides and Clostridial genera, representatives of the Bacteroidetes and Firmicutes phyla establish a new steady state after about one month approximating the composition of the conventional microbiota in both humans and mice where about 90% of the bacteria taxa reside within these two phyla.
  • Clostridia may be due to a syntrophic synergism between these two genera that increases the richness of the gut microbiota and helps to both stabilize and enhance resiliency of the ASF community long-term as observed over the course of a four months study.
  • the impact of the transplanted ASF community on host metabolism was also evaluated.
  • the gut microbiota and its host co-existed in a state of mutualism that, in part, involves interactions involving nutrition and metabolism.
  • the gut microbiota whose collective genome exceeded its mammalian host by about 150-fold, exhibited functionalities that serve as an example of collective "superorganism".
  • glycan degrading genes in the microbiome responsible for the fermentation of complex carbohydrates with the production of short chain fatty acids, compounds that have important functions in host immunity and metabolism.
  • these results demonstrated a syntrophic co-metabolic interaction between a mammalian host and its microbiota to maintain nitrogen balance dependent upon the microbial urease gene, a functionality not present in the mammalian genome.
  • a waste product of the mammalian host, urea is delivered to the colon where the gut microbiota scavenges nitrogen by hydrolyzing urea into carbon dioxide and ammonia, the latter of which is utilized by the gut microbiota for protein synthesis, reabsorbed by the host, or excreted in the feces.
  • Example 7 A single bacterial species lacking a urease gene can significantly reduce fecal ammonia levels when inoculated into a properly prepared host.
  • a murine commensal E. coli strain (MP1) tagged with GFP known to colonize mice ( Lasaro, M., et al. J. Bacteriol. 196:1723-32 (2014 )that lacks a urease gene and shows no urease activity, was inoculated by daily oral gavage for 5 days (1 X 10 9 cfu) into mice pretreated with vancomycin, neomycin, and PEG. Colony counts revealed long-term colonization of this bacterium at a level of approximately 1 X 10 6 cfu/gram of feces. Quantification of fecal ammonia levels revealed that fecal ammonia was reduced significantly by approximately 6-fold after 50 days.
  • Example 8 Procedure for designing human bacterial communities for treating hyperammonemia and other forms of dysbiosis.
  • the altered gut micriobiota described herein is useful for reducing urease production from the gut.
  • additional novel defined microbial consortia can be obtained using the following exemplary steps. Stool is collected from adults in robust health who meet the following criteria and none of the following.
  • bacterial strains are purified by repeated rounds of streaking and limiting dilution under anaerobic conditions on multiple types of media. They are then tested for survival at after freezing at -80°C and subsequent thawing.
  • information on Genus and species attribution is used to ascertain the probability of containing urease genes by comparison to databases of complete bacterial genome sequences.
  • Suitable strains are then formulated into communities with structures resembling resilient human communities from healthy individuals (containing multiple Firmicutes, Bacteriodetes, and other taxa), but using only strains identified as lacking urease genes.
  • Human strains are also evaluated by comparison to murine ASF. Specifically, because Parabacteriodes was predominant in ASF in colon, this approach will ensure that members of the Parabacteriodes/Bacteriodes group are represented in engineered communities.
  • consortia Following identification of suitable consortia, these are tested in mice for ability to persist in a suitably prepared animal. The newly identified consortia can then be assess for alteration of the disease parameters targeted for alleviation. For example, in the present case, the consortia are tested for ability to lower fecal ammonia level and/or in rescue in models of ammonia toxicity.
  • Selected bacterial strains are characterized by near-complete genome sequencing and the sequences are assessed for the presence or absence certain types of genes, e.g., urease genes, antibiotic resistance genes, and or virulence gene representation. Once satisfied that the strains meet these criteria, strains can be tested functionally for ureases activity and/or for antibiotic resistance to identify antibiotic classes to which all strains are sensitive. Should any adverse gene content be identified, the consortia will be edited to remove any members with adverse gene content or activities as set forth above, then the final community retested.
  • genes e.g., urease genes, antibiotic resistance genes, and or virulence gene representation.
  • this community can be used to advantage in human subjects in the methods of treatment described herein.

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Claims (14)

  1. Zusammensetzung, umfassend ein Konsortium von Bakterien, die kein Urease-Gen enthalten oder keine Urease-Aktivität aufweisen, zur Verwendung in einem Verfahren zur Behandlung von Hyperammonämie, Leberenzephalopathie oder einer anderen Leberstörung, charakterisiert durch Hyperammonämie, in einem Individuum mit Bedarf daran, wobei
    a) dem Individuum vor Verabreichen der Zusammensetzung eine wirksame Menge eines oder mehrerer Antibiotika verabreicht wird, die ausreicht, um die Population von Bakterien in der Darmflora auf ein Niveau zu senken, das für die Repopulation von neu eingeführten Bakterien geeignet ist; und wobei
    b) dem Individuum vor Verabreichen der Zusammensetzung weiters eine wirksame Menge eines Abführmittels verabreicht wird, wobei das Verfahren Folgendes umfasst:
    c) Verabreichen der Zusammensetzung an das Individuum unter Bedingungen, unter denen die Bakterien den Darm besiedeln, wobei die Zusammensetzung die Bakterien Paraprevotella clara, Bifidobacterium longum, Collinsella aerofaciens, Coprococcus comes, Dorea longicatena, Bacteroides eggerthii str. und Bacteroides vulgates umfasst.
  2. Zusammensetzung zur Verwendung nach Anspruch 1, wobei das Abführmittel aus der aus Polyethylenglykol (PEG), Magnesiumcitrat, Natriumpicosulfat plus Magnesiumcitrat, PEG plus Ascorbinsäure und Natriumphosphat bestehenden Gruppe ausgewählt ist.
  3. Zusammensetzung zur Verwendung nach Anspruch 1, wobei die Verabreichung der Zusammensetzung über eine Route erfolgt, die aus der aus durch Endoskopie, durch Enteroskopie, durch Koloskopie, einen Nasoduodenalkatheter, einem Einlauf und oral in einer einnehmbaren Kapsel oder Pille bestehenden Gruppe ausgewählt ist.
  4. Zusammensetzung zur Verwendung nach Anspruch 1, wobei ein oder mehrere Antibiotika für zumindest etwa 48, 72 oder 96 h vor der Verabreichung der Zusammensetzung verabreicht werden und wobei das Antibiotikum Neomycin und Vancomycin ist.
  5. Zusammensetzung zur Verwendung nach Anspruch 1, wobei etwa 12 bis 36 h vor der Verabreichung der Zusammensetzung eine Darmspülung durchgeführt wird.
  6. Zusammensetzung zur Verwendung nach Anspruch 5, wobei die Zusammensetzung weiters einen oder mehrere Bakterienstämme umfasst, die einem Genus angehören, das aus der aus Bifidobacterium, Bacteroides, Tannerella, Parabacteroides, Bacillus, Lactobacillus, Anaerostipes, Blautia, Coprococcus, Dorea, Clostridium XI, Collinsella und Paraprevotella bestehenden Gruppe ausgewählt ist, oder wobei das definierte mikrobielle Konsortium aus Paraprevotella clara, Bifidobacterium longum, Collinsella aerofaciens, Coprococcus comes, Dorea longicatena, Bacteroides eggerthii str. und Bacteroides vulgates besteht oder wobei das Konsortium weiters Clostridium sp., Lactobacillus sp., Lactobacillus murinus, Mucispirillum schaedleri, Eubacterium plexicaudatum, Firmicutes-Bakterium, Clostridium sp. und Parabacteroides sp. umfasst.
  7. Zusammensetzung zur Verwendung nach Anspruch 1, wobei die Erkrankung eine Stoffwechselstörung ist, die aus der aus Hyperammonämie, transienter Hyperammonämie bei Neugeborenen oder Leberenzephalopathie bestehenden Gruppe ausgewählt ist.
  8. Zusammensetzung zur Verwendung nach Anspruch 1, wobei die Behandlungseffizienz weiters durch Bewerten eines Parameters überwacht wird, der aus der aus 16S-rRNA-Kopienanzahl, 16S-rRNA-Gensequenzierung, Urease-Werten im Stuhl; zirkulierenden oder im Stuhl vorhandenen Aminosäuren, biogenen Aminen, Ammoniakwerten im Stuhl und zirkulierenden Ammoniakwerten bestehenden Gruppe ausgewählt ist.
  9. Zusammensetzung zur Verwendung nach Anspruch 8, wobei die 16S-rRNA-Kopienanzahl vor der Verabreichung der Zusammensetzung um zumindest etwa 2 bis 4 log reduziert wird.
  10. Zusammensetzung, umfassend ein Konsortium von Bakterien, die kein Urease-Gen enthalten oder keine Urease-Aktivität aufweisen, umfassend die Bakterien Paraprevotella clara, Bifidobacterium longum, Collinsella aerofaciens, Coprococcus comes, Dorea longicatena, Bacteroides eggerthii str. und Bacteroides vulgates, wobei das Konsortium in einem biologischen Träger vorhanden ist, der für die Verabreichung an den Darm und die Besiedelung des Darms geeignet ist.
  11. Zusammensetzung nach Anspruch 10, wobei die Zusammensetzung zumindest ein weiteres Bakterium umfasst, das kein Urease-Gen enthält oder keine Urease-Aktivität aufweist, das aus Bifidobacterium, Bacteroides, Tannerella, Parabacteroides, Bacillus, Lactobacillus, Anaerostipes, Blautia, Coprococcus, Dorea und Clostridium XI ausgewählt ist.
  12. Zusammensetzung, umfassend eine einzelne Bakterienspezies, die kein Urease-Gen enthält oder keine Urease-Aktivität aufweist, zur Verwendung in einem Verfahren zur Behandlung von Hyperammonämie, Leberenzephalopathie oder einer anderen Leberstörung, gekennzeichnet durch Hyperammonämie, in einem Individuum mit Bedarf daran, wobei
    a) dem Individuum vor der Verabreichung der Zusammensetzung eine wirksame Menge Vancomycin und Neomycin verabreicht wird, die ausreichend ist, um die Population von Bakterien in der Darmflora auf ein Niveau zu senken, das für die Repopulation des neu eingeführten Bakteriums geeignet ist; und wobei
    b) dem Individuum vor der Verabreichung der Zusammensetzung weiters eine wirksame Menge eines Abführmittels verabreicht wird; wobei das Verfahren Folgendes umfasst:
    c) das Verabreichen der Zusammensetzung an das Individuum unter Bedingungen, unter denen das Bakterium den Darm besiedelt.
  13. Zusammensetzung zur Verwendung nach Anspruch 12, wobei das Bakterium aus der aus Urease-negativem Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus lactis, Lactobacillus dubrueckii bulgaricus, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium breve, E. coli Nissle und Streoptococcus thermopiles bestehenden Gruppe ausgewählt ist.
  14. Zusammensetzung zur Verwendung nach Anspruch 1 oder 12, wobei die Zusammensetzung die Bakterienspezies in Verdünnung in einer biologisch kompatiblen Lösung aufweist, die gegebenenfalls Exzipienten enthält, die für die Verabreichung an das Individuum geeignet sind.
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